Reflected fluorescence microscope with multiple laser and excitation light sources

ABSTRACT

Caged reagent release experiments are carried out by introducing laser light of a laser light source for the Caged reagent release of a first reflected illumination optical system light path into a sample side through a first dichroic mirror on a cube turret and also introducing excitation light of a mercury lamp for the excitation light irradiation of a third reflected illumination optical system light path into the sample side through a first dichroic mirror on a slider. Laser trap experiments are carried out by introducing excitation light of a mercury lamp for the excitation light irradiation of a second reflected illumination optical system light path into the sample side through a second dichroic mirror on the cube turrent and also introducing laser light from the laser light source for the laser trap of the third reflected illumination optical system light path into the sample side through a second dichroic mirror on the slider.

BACKGROUND OF THE INVENTION

The present invention relates to a reflected fluorescence microscope,and more particularly to a reflected fluorescence microscope employingthe laser trap method or the Caged reagent release method used as ameans for cellular operation in the cell physiological field.

The reflected fluorescence observation using a reflected fluorescencemicroscope is now a method widely and generally employed as a method ofmorphologic observation of specific substances inside cells in thebiological field.

Recently, the laser trap method or the Caged reagent release method hasbeen widely employed as an operating intracellular substances operatingmethod using a reflected illumination means of a microscope,particularly, in the cell physiological field.

The laser trap method (light pin set method), which is a method ofapplying laser light to an arbitrary substance in a sample and opticallycapturing the substance, is employed as a means for measuring energywhen protein in cells moves. Infrared laser light is used for theapplying means. On the other hand, the Caged reagent release method is amethod of bonding a Caged reagent with molecules having physiologicalactivity, administrating it into cells in a state where activity isrestricted, and releasing the bonding by applying light thereto so as torecover the activity of the cells. Ultraviolet light is used for theapplying means.

As described above, a reflected fluorescence microscope employing thelaser trap method or the Caged reagent release method needs variouslight sources for cellular operation other than the general reflectedfluorescence illumination.

In a conventionally used general reflected fluorescence microscope, asshown in FIG. 1, an excitation light source 104 for fluorescenceobservation is provided for a first dichroic mirror DM1 providedtogether with a barrier filter BA in an observation optical system lightpath 103 connecting an object lens 101 and an eyepiece 102, a seconddichroic mirror DM2 is provided between the first dichroic mirror DM1and the excitation light source 104, a band pass filter BP is providedbetween the second dichroic mirror DM2 and the excitation light source104, and a light source 105 for sample operation is provided in a lightpath which is orthogonal at the second dichroic mirror DM2.

Further, Jpu. Pat. Appln. KOKAI Publication No. 8-234110 discloses areflected fluorescence microscope, as shown in FIG. 2, wherein a firstdichroic mirror DM1 for introducing excitation light from an excitationlight source 108 for fluorescence observation into an observationoptical system light path 107, and a second dichroic mirror DM2 forintroducing laser light of the laser trap from a laser light source 109thereto are provided at two stages.

In the prior art shown in FIG. 1, however, not only the band pass filterBP, but also the first dichroic mirror DM1 need to be exchanged ifexecuting the fluorescence observation by switching the excitationwavelength of the excitation light source 104 is considered. At thistime, the first dichroic mirror DM1 and the band pass filter BP, whichare provided separately from one another, must be exchanged separately,and the work for this exchange is complicated and troublesome.

On the other hand, in the prior art in FIG. 2 disclosed in Jpn. Pat.Appln. KOKAI Publication No. 8-234110, in a case where the firstdichroic mirror DM1 is provided more closely to the sample side than thesecond dichroic mirror DM2, if, for example, the laser wavelength rangeof the laser light source 109 is changed to the ultraviolet range, whichis to be used for the Caged reagent release, the first dichroic mirrorDM1 and the barrier filter BA must have a characteristic of transmittingthe last wavelength from the laser light source 109 in order tocertainly introduce the laser light reflected at the second dichroicmirror DM2 into the sample side. At every introduction of the laserlight, a troublesome work such as exchange of the first dichroic mirrorDM1 and the barrier filter BA to optimal ones need to be carried outand, therefore, there is little realizability of this system Actually,the laser light source 109 cannot be applied to the object of use in therange other than the infrared range for the laser trap and can be hardlyused for a general purpose Further, since the second dichroic mirror DM2is fixed in the observation optical system light path 107, no problemsoccur in a general fluorescence observation, but the fixation is areason for loss of light amount in the tiny fluorescence observationsuch as measurement of light at the one-molecule level and is nottherefore preferable.

BRIEF SUMMARY OF THE INVENTION

Accordingly, it is an object of the present invention to provide areflected fluorescence microscope having a plurality of light paths inthe reflected illumination optical system, which are not limitedrespectively in use, and being largely usable for a general purpose.

According to one aspect of the present invention, there is provided areflected fluorescence microscope for allowing a plurality of reflectedillumination light beams to be selectively irradiated onto a sample, tocarry out optical operations of the sample and its fluorescenceobservation simultaneously, the microscope comprising: a first reflectedillumination optical system light path provided in a direction whichcrosses an observation optical system light path to the sample at apoint of intersection; a second reflected illumination optical systemlight path provided in a direction which crosses the observation opticalsystem light path at the same point of intersection as that between theobservation optical system light path and the first reflectedillumination optical system light path; and a third reflectedillumination optical system light path provided in a direction whichcrosses the observation optical system light path at a different pointof intersection from that of the first or second reflected illuminationoptical system light path.

The microscope may further comprise wavelength discriminating meansprovided to be inserted or detached at the point of intersection betweenthe observation optical system light path and the third reflectedillumination optical system light path.

The microscope may further comprise a fourth reflected illuminationoptical system light path provided in a direction which crosses theobservation optical system light path at a different point ofintersection from that of the first, second or third reflectedillumination optical system light path.

According to another aspect of the present invention, there is provideda reflected fluorescence microscope for allowing a plurality ofreflected illumination light beams to be selectively irradiated onto asample, to carry out optical operations of the sample and itsfluorescence observation simultaneously, the microscope comprising: afirst reflected illumination optical system light path provided in adirection which crosses an observation optical system light path to thesample at a point of intersection; a second reflected illuminationoptical system light path provided in a direction which crosses theobservation optical system light path at the same point of intersectionas that between the observation optical system light path and the firstreflected illumination optical system light path; a first movable memberhaving a cube including a first dichroic mirror and a cube including asecond dichroic mirror, for providing a desired one of the cubes to beinserted or detached at the point of intersection between theobservation optical system light path and the first or second reflectedillumination optical system light path; a third reflected illuminationoptical system light path provided in a direction which crosses theobservation system light path at a different point of intersection fromthat between the first or second reflected illumination optical systemlight path; and a second movable member having a cube including a thirddichroic mirror, for providing the cube to be inserted or detected atthe point of intersection between the observation optical system lightpath.

In the microscope, the first dichroic mirror may reflect the light fromthe first reflected illumination optical system light path to a side ofthe sample, while transmitting fluorescence emitted from the sample. Thesecond dichroic mirror may reflect the light from the second reflectedillumination optical system light path to a side of the sample, whiletransmitting fluorescence emitted from the sample. The third dichroicmirror may reflect the light from the third reflected illuminationoptical system light path to a side of the sample, while transmittingfluorescence emitted from the sample.

In the microscope, the first movable member may be a cube turret. Thesecond movable member may be a slider.

The microscope may further comprise a laser light source for forming thefirst reflected illumination optical system light path. The microscopemay further comprise a mercury light source for forming the secondreflected illumination optical system light path. The microscope mayfurther comprise at least one of a laser light source and a mercurylight source for forming the third reflected illumination optical systemlight path.

The microscope may further comprise a fourth reflected illuminationoptical system light path provided in a direction which crosses thethird reflected illumination optical system light path at a point ofintersection. In this case, the microscope may further comprise a sliderfor providing a total reflection mirror to be inserted or detached at apoint of intersection between the third and fourth reflectedillumination optical system light paths.

Additional objects and advantages of the invention will be set forth inthe description which follows, and in part will be obvious from thedescription, or may be learned by practice of the invention. The objectsand advantages of the invention may be realized and obtained by means ofthe instrumentalities and combinations particularly pointed outhereinafter.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING

The accompanying drawings, which are incorporated in and constitute apart of the specification, illustrate presently preferred embodiments ofthe invention, and together with the general description given above thedetailed description of the preferred embodiments given below, serve toexplain the principles of the invention.

FIG. 1 is a diagram illustrating a schematic structure of an example ofa conventional reflected fluorescence microscope;

FIG. 2 is a diagram illustrating a schematic structure of anotherexample of a conventional reflected fluorescence microscope,

FIG. 3 is a diagram illustrating a schematic structure of a firstembodiment of the present invention.

FIG. 4 is a diagram illustrating the schematic structure of the firstembodiment of the present invention in a different mode of operationthan the one in FIG. 3;

FIG. 5 is a graph showing spectral transmittance characteristics of adichroic mirror DM1 used in the first embodiment of the presentinvention;

FIG. 6 is a graph showing spectral transmittance characteristics of adichroic mirror DM2′ used in the first embodiment of the presentinvention;

FIG. 7 is a graph showing spectral transmittance characteristics of adichroic mirror DM2, an exactuation filter EX2, and a barrier filter BA2used in the first embodiment of the present invention;

FIG. 8 is a graph showing spectral transmittance characteristics of adichroic mirror DM1′, and excitation filter EX1′, and a barrier filterBA1′ used in the first embodiment of the present invention;

FIG. 9 is a diagram illustrating a schematic structure of a secondembodiment of the present invention;

FIG. 10 is a diagram illustrating a partially modified schematicstructure of the second embodiment of the present invention;

FIG. 11 is a diagram illustrating a schematic structure of a thirdembodiment of the present invention;

FIG. 12 is a diagram illustrating a schematic structure of a fourthembodiment of the present invention; and

FIG. 13 is a diagram illustrating a schematic structure of a firthembodiment of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

Embodiments of the present invention will be described below withreference to the drawings.

1st Embodiment

FIGS. 3 and 4 illustrate a schematic structure of an inverted-typereflected fluorescence microscope to which the present invention isapplied. In the figures, reference numeral 1 denotes a sample. An objectlens 2 is provided under the sample 1, and a cube turret CT asrepresented in a broken line is provided in an optical axis 31 of theobservation optical system light path passing through the object lens 2.

The cube turret CT includes a cube CV1 which has a dichroic mirror DM1with an inclination angle of 45 degrees to the optical axis 31, and acube CV1′ which has a dichroic mirror DM1′ with an inclination angle of45 degrees to the optical axis 31, a barrier filter BA1′ and anexcitation filter EX1′ The cube turret CT allows the cube CV1 or CV1′ tobe selectively positioned at a point O1 which the optical axis 31passes, by rotating around a rotary axis (not shown) in a planeperpendicular to the optical axis 31.

The cube turret CT shown in FIG. 3 includes only two cubes. However, atleast four cubes having the same structure and shape as those of thecubes CV1 and CV1′ can be inserted or detached, and reflective surfaceof a dichroic mirror of an arbitrary cube can be positioned at the pointO1 at which the optical axis 31 passes, by the rotation of the turretand a positioning mechanism (not shown). For example, FIG. 4 illustratesan example in which a cube CV1″ having a dichroic mirror DM1″ and abarrier filter BA1″ is included in the cube turret CT, and the cubeturret CT is constituted to be applicable to a case where thewavelengths of a laser light source 3 and a mercury light source 6described later are varied. The cube CV1′ holds the dichroic mirror DM1′so that the reflective surface of the dichroic mirror DM1′ faces themercury light source 6 described later when the optical axis 31 passesthrough the cube CVI′.

A relay lens 5, a collector lens 4 and a laser light source 3 areprovided in a common straight line, in an optical axis 32 of a firstreflected illumination optical system light path which extends from thepoint O1 of he cube turret CT, perpendicularly to the optical axis 31.In addition, a relay lens 8, a collector lens 7 and a mercury lightsource 6 are provided in a common straight line, in an optical axis 33of a second reflected illumination optical system light path whichextends from the point O1, perpendicularly to both the optical axis 31and the direction extending from the point O1 to the laser light source3.

A slider SV represented in a broken line, which is described later, isprovided to be movable along an optical axis 34 under the cube turretCT.

On the slider SV, a cube CV2 including a dichroic mirror DM2 having anangle of inclination at 45 degrees to the optical axis 32, a barrierfilter BA2 and an excitation filter EX2, an a cube CV2′ including adichroic mirror DM2′ provided parallel to the dichroic mirror DM2, arealigned in a direction in which the optical axis 32 is reflected by thedichroic mirror DM2 (or in the direction of the optical axis 34). Thecubes CV2 and CV2′ can be inserted into or detached from the slider SV.A condense lens 15 is provided just under the cube CV2 of the slider SV,and a condense lens 16 is provided just under the cube CV2′.

The slider SV moves along the direction of the optical axis 34, so thatthe reflective surface of the dichroic mirror DM2 of the cube CV2 or thereflective surface of the dichroic mirror DM2′of the cube CV2′ can beselectively positioned at the point O2 at which the optical axis 31passes.

The switch stopping position of the dichoric mirrors DM2 and DM2′ on theslider SV is set by a positioning mechanism (not shown)

A relay lens 11, a collector lens 10 and a mercury light source 9 areprovided in the optical axis 34 of a third reflected illuminationoptical system light path reflected from the point O2 of the slider SV.A slider SV′ is provided to be inserted or detached, at a point O3 atwhich the optical axis 34 passes, in the optical axis 34 extendingbetween the relay lens 11 and the collector lens 10, and a collectorlens 13 and a laser light source 12 are provided perpendicularly to theoptical axis 34 via the slider SV′.

In this case, a total reflection mirror 14 is provided on the sliderSV′. By moving the slider SV′ in a direction represented by an arrow inthe figure, the refelective surface of the total reflection mirror 14can be inserted or detached at the point O3 and a luminous flux from thelaser light source 12 can be introduced toward the point O2 from whichthe optical axis 31 extends. The inserted and detached position of theslider SV′ is automatically set by a positioning mechanism (not shown).

On the other hand, a half prism 17 for splitting a light path and atotal reflection mirror 18 for introducing the optical axis 31 into aneyepiece section are provided, in the optical axis 31, under the sliderSV.

The observation light transmitted through the condense lens 15 or 16 issplit into two beams by the beam splitter 17. The light beam reflectedby the beam splitter 17 is introduced into a photographing light pathand forms an image on a light receiving surface Ph of a photographingdevice or a TV camera.

The light beam transmitted through the beams spitter 17 is reflected bythe total reflection mirror 18 and introduced into an observation lightpath. After an image is formed at an intermediate imaging point A in theobservation light path, it is relayted by a relay lens R1 and formedagain at a point B This image is observed through an eyepiece Oc.

Next, operations of the embodiment thus constituted will be explained.

It is assumed here that an N₂ laser having a wavelength of 337 nm forthe Caged reagent release is used as the laser light source 3, mercurylight to take out the light having a wavelength of 488 nm for thefluorescent observation, for irradiation of the excitation light, isused as the mercury light source 6, mercury light to take out the lighthaving a wavelength of 532 nm for the flourescent observation, forirradiation of the excitation light, is used as the mercury light source9, and an IR laser having a wavelength of 1064 nm for the laser trap isused as the laser light source 12.

As shown in FIG. 3, first, the cube CV1 of the cube turret CT ispositioned in the optical axis 31 and the cube CV2 of the slider SV isinserted into the optical axis 31 of the observation optical systemlight path. Then the slider SV′ is removed from the optical axis 34.

In this state, the N₂ laser for the Caged reagent release having awavelength of 337 nm from the laser light source 3 is made incident onthe dichroic mirror DM1 of the cube CV1 via the collector lens 4 andrelay lens 5. If the dichroic mirror DM1 having the spectraltransmittance characteristics of reflecting the light having awavelength of less than 400 nm and transmitting the light having awavelength exceeding 400 nm as shown in FIG. 5, is used, the N₂ laser ofthe laser light source 3 is reflected at the dichroic mirror DM1 andirradiated at a predetermined position in the observation visual view ofthe sample 1 through the object lens 2 so as to carry out the Cagedreagent release.

On the other hand, in this state, the dichroic mirror DM2 of the cubeCV2 of the slider SV is positioned at the point O2 at which the opticalaxis 31 passes, and the slider SV′ is detached from the optical axis 34.Therefore, the mercury light of the mercury light source 9 having awavelength of 532 nm is made incident on the dichroic mirror DM2′ viathe collector lens 10 and the relay lens 11. If the dichroic mirror DM2′having the spectral transmittance characteristics of transmitting thelight having a wavelength of less than 800 nm and reflecting the lighthaving a wavelength exceeding 800 nm as shown in FIG. 6, is used here,the light from the mercury light source 9 is transmitted through thedichroic mirror DM2′ and made incident on the excitation filter EX2 ofthe cube CV2. Further, if the excitation filter EX2 having the spectraltransmittance characteristics of transmitting the light having anexcitation light wavelength ranging from 510 nm to 550 nm as shown inFIG. 7, is used, only the excitation light is transmitted and madeincident on the dichroic mirror DM2. Moreover, if the dichroic mirrorDM2 having the spectral transmittance characteristics of reflecting thelight having a wavelength of less than 570 nm and transmitting the lighthaving a wavelength exceeding 570 nm as shown in FIG. 7, is used, theexcitation light from the excitation filter EX2 is reflected upward atthe dichroic mirror DM2, transmitted through the dichroic mirror DM1 andirradiated in the observation visual view of the sample 1 through theobject lens 2.

When fluorescence is emitted from the sample 1 by irradiation of theexcitation light, the fluorescence is made incident on the barrierfilter BA2 of the cube CV2 through the dichroic mirrors DM1 and DM2after passing through the object lens 2. If the barrier filter BA2having the spectral transmittance characteristics of reflecting thelight having a wavelength of less than 590 nm and transmitting the lighthaving a wavelength exceeding 590 nm as shown in FIG. 7, is used here,the fluorescence is transmitted and then observed through the half prism17 and the total reflection mirror 18.

The Caged reagent release and the fluorescence observation can becarried out simultaneously.

The characteristics of the dichroic mirrors DM1 and DM2 used when theCaged reagent release and the fluorescence observation are carried outsimultaneously will be summarized as follows.

That is, the dichroic mirror DM1 reflects the laser light from the laserlight source 3 (having a wavelength of less than 400 nm), and transmitsthe excitation light from the mercury light source 9 and the light(having a wavelength exceeding 400 nm) emitted from the sample 1 (seeFIG. 5). The dichroic mirror DM2 reflects the excitation light from themercury light source 9 (having a wavelength of less than 570 nm), andtransmits the light (having a wavelength exceeding 570 nm) emitted fromthe sample 1 (see FIG. 7).

Next, as shown in FIG. 4, the cube turret CT is rotated in a directionrepresented by an arrow to position the cube CV1′ in the optical axis31, the cube CV2′ is inserted into the optical axis 31 by switching theslider SV, and the total reflection mirror 14 is inserted into theoptical axis 34 by switching the slider SV′.

In this state, the mercury light for observation of the fluorescencehaving a wavelength of 488 nm, from the mercury light source 6, is madeincident on the excitation filter EX1′ of the cube CV1′ through thecollector lens 7 and the relay lens 8. If the excitation filter EX1′having the spectral transmittance characteristics of transmitting thelight having a wavelength ranging from 470 nm to 490 nm as shown in FIG.8, is used here, only the excitation light is made incident on thedichroic mirror DM1′. Further, if the dichroic mirror DM1′ having thespectral transmittance characteristics of reflecting the light having awavelength of less than 500 nm and transmitting the light having awavelength exceeding 500 nm as shown in FIG. 8, is used here, theexcitation light of the excitation filter EX1′ is reflected upward atthe dichroic mirror DM1′ and irradiated into the observation visualfield of the sample 1 through the object lens 2.

When the fluorescence is emitted from the sample 1 by irradiation of theexcitation light, this fluorescence is transmitted through the dichroicmirror DM1′ after passing through the object lens 2, and is madeincident on the barrier filter BA1′ of the cube CV1′. If the barrierfilter BA1′ having the spectral transmittance characteristics ofreflecting the light having a wavelength of less than 515 nm andtransmitting the light having a wavelength exceeding 515 nm as shown inFIG. 8, is used here, the fluorescence is transmitted and is madeincident on the dichroic mirror DM2′ of the slide SV. Since thewavelength of the fluorescence at this time is less than 800 nm, thefluorescence is transmitted through the dichroic mirror DM2′ andobserved through the half prism 17 and the total reflection mirror 18.

On the other hand, since the slider SV′ is inserted into the opticalaxis 34, the IR laser for the laser trap having a wavelength of 1064 nmfrom the laser light source 12 is made incident on the total reflectionmirror 14 after passing through the collector lens 13 to be totallyreflected at the total reflection mirror 14, and is made incident on thedichroic mirror DM2′ of the cube CV2′. Then, the laser beam is reflectedupward by the spectral transmittance characteristics of the dichroicmirror DM2′ and is made incident on the barrier filter BA1′ of the cubeCV1′. The laser beam is transmitted by the spectral transmittancecharacteristics of the barrier filter BA1′, further transmitted by thespectral transmittance characteristics of the dichroic mirror DM1′, andmade incident on the object lens 2.

The laser light beam incident on the object lens 2 is collected, andirradiated to a predetermined position on the sample 1 as a laser spot.Fine substances in the sample 1 can be optically captured near theirradiated laser collection point.

By these operations, capture of the fine substances, measurement of atiny force, etc. can be simultaneously carried out by the observation offluorescence and the laser trap.

The characteristics of the dichroic mirrors DM1′ and DM2′ used when thecapture of fine substances, measurement of a tiny force, etc. based onthe observation of fluorescence and the laser trap are carried out, canbe summarized as follows.

That is, the dichoric mirror DM1′ reflecting the excitation light(having a wavelength of less than 500 nm) from the mercury light source6 bans also transmitting the laser light transmitted from the laserlight source 12 and the light (having a wavelength exceeding 500 nm)emitted from the sample 1, is used (see FIG. 8). In addition, thedichroic mirror DM2′ reflecting the laser light (having a wavelength ofmore than 800 nm) from the laser source 12 and transmitting the light(having a wavelength less than 800 nm) emitting from the sample 1, isused (see FIG. 6).

With this structure, the experiments of the Caged reagent releaseexperiment based on the Caged reagent release and the observation of thefluorescence can be carried out, by introducing the laser light from thelaser light source 3 for the Caged reagent release of the firstreflected illumination optical system light path into the sample 1 sidethrough the dichroic mirror DM1 of the cube CV1, and simultaneouslyintroducing the excitation light from the mercury lamp 9 for irradiationof the excitation light of the third reflected illumination opticalsystem light path into the sample 1 side through the dichroic mirrorDM2′ of the cube CV2′ and dichroic mirror DM2 of the cube CV2. On theother hand, the laser trap experiments such as capture fine substances,measurement of a tiny force, etc. based on observation of thefluorescence and the laser trap can be selectively carried out, byintroducing the excitation light from the mercury lamp 6 for excitationlight irradiation of the second reflected illumination optical systemlight path to the sample 1 side through the dichroic mirror DM1′ of thecube CV1′, and simultaneously introducing the laser light from the laserlight source 12 for the laser trap of the third reflected illuminationoptical system light path to the sample 1 side through the dichroicmirror DM2′ of the cube CV2′. That is, these two experiments can beselectively carried out together with the sample operation, and areflected fluorescence microscope usable for a general purpose can beprovided.

An inverted-type microscope has been described in the above explanationof the first embodiment, but the present invention can be applied to ageneral erecting-type microscope. The kind, wavelength, number andarrangement of the light sources used at this time may not be the same.The crossing angle between each illumination optical system light pathand the observation optical system light path (optical axis 31) does notneed to be set at 90 degrees, and the angles may be different from oneanother Further, the characteristics of the dichroic mirrors used atthis time may not be the same, and a dichroic prism or other suitablewavelength classifying means can be applied if it is suitable todesirable luminous flux wavelength characteristics. The mechanism ofinserting the dichroic mirror into the optical axis or detaching ittherefrom may not be the same as the cube turret CT or the slider SV,and any other mechanism can be used. One of the condense lenses 15 and16 may be fixed at a predetermined point in an optical axis of the mainbody of the microscope, without attaching both of the condense lenses 15and 16 to the slider SV.

2nd Embodiment

FIG. 9 illustrates a schematic structure of a second embodiment of thepresent invention, and the same portions as those seen in FIG. 3 areindicated by the same reference numerals.

In the case shown in FIG. 9, the laser light source 12, the collectorlens 13, the total reflection mirror 14, the slider SV′, the dichroicmirror DM2′ and the cube CV2′ are removed from the structure of FIG. 3,and the other portions are the same as FIG. 3.

That is, the laser light source 12 and the cube CV2′ are removed fromthe structure of FIG. 3, in the second embodiment thus constituted.

For this reason, if unnecessary optical elements are not inserted in theparallel luminous flux light path of the observation optical system(i.e. between the object lens 2 and the condense lens 16) by switchingthe slider SV′ to insert the condense lens 16 into the optical axis 31,and rotating the turret of the cube turret CT to position the portionincluding no cubes to the optical axis 31 or detach the cube CV1, thepresent invention can be switched to a general inverted-type microscopeby only simple operations.

With these operations, by removing from the observation light path thedichroic mirrors or barrier filters which are not used in the generalfluorescence observation of irradiating only a single excitation lightbeam, unexpected inconvenience that the fluorescence of the wavelengthband in a certain range may be reflected and removed from theobservation light path, can be prevented in advance since the unusedcube is in the observation light path. In addition, by removingunnecessary optical elements, losing the light amount at observation ofthe fine fluorescence can be prevented in advance, and deterioration ofan observation image obtained by combining the fluorescence observationand the other microscopic method can be prevented.

If the laser trap is employed in the structure shown in FIG. 9, the cubeCV2 of the slider SV shown in FIG. 9 may be replaced with the cube CV2′as shown in FIG. 10, and the laser light source 12 may be used insteadof the mercury light source 9.

With this structure, the laser trap and the fluorescence observationscan be carried out simultaneously, and the same advantage as theabove-described one can be expected. In this embodiment, the cubeattached to the slider SV may be replaced with the other one inaccordance with the light source which is to be used.

An inverted-type microscope has been described in the above explanationof the second embodiment, but the present invention can be applied to ageneral erecting-type microscope. The kind, wavelength, number andarrangement of the light sources used at this time may not be the same.The crossing angle between each illumination optical system light pathand the observation optical system light path (optical axis 31) does notneed to be set 90 degrees, and the angles may be different from oneanother. Further, the characteristics of the dichroic mirrors used atthis time may not be the same, and a dichroic prism or other suitablewavelength classifying means can be applied if it is suitable todesirable luminous flux wavelength characteristics. The mechanism ofinserting the dichroic mirror into the optical axis or detaching ittherefrom may not be the same as the cube turret (T or the slider SV,and any other mechanism can be used. One of the condense lenses 15 and16 may be fixed at a predetermined point in an optical axis of the mainbody of the microscopes, without attaching both of the condense lenses15 and 16 to the slider SV.

3rd Embodiment

FIG. 11 illustrates a schematic structure of a third embodiment of thepresent invention, and the same portions as those seen in FIG. 3 areindicated by the same reference numerals.

In the case shown in FIG. 11, a slider SV″ newly including a condenselens 19, which is formed by extending the slider SV, is provided insteadof the slider SV shown in FIG. 3. The other portions are the same asFIG. 3.

In the third embodiment thus constituted, a positioning mechanism (notshown) allows the cubes CV2 and CV2′, and the condense lens 19 to beinserted into the parallel luminous flux light path of the observationoptical system by switching the slider SV″.

With this operation, the above-described advantage of the secondembodiment can be obtained without losing any advantage of the firstembodiment. That is, various luminous flux can be selectively used, andan inverted-type microscope which is usable in a general manner can beprovided, only by the simple switching.

An inverted-type microscope has been described in the above explanationof the third embodiment, but the present invention can be applied to ageneral erecting-type microscope. The kind, wavelength, number andarrangement of the light sources used at this time may not be the same.The crossing angle between each illumination optical system light pathand the observation optical system light path (optical axis 31) does notneed to be set at 90 degrees, and the angles may be different from oneanother Further, the characteristics of the dichroic mirrors used atthis time may not be the same, and a dichroic prism or other suitablewavelength classifying means can be applied if it is suitable todesirable luminous flux wavelength characteristics. The mechanism ofinserting the dichroic mirror into the optical axis or detaching ittherefrom may not be the same as the cube turret CT or the slider SV,and any other mechanism can be used. One of the condense lenses 15, 16and 19 may be fixed at a predetermined point in an optical axis of themain body of the microscope, without attaching all of the condenselenses 15, 16 and 19 to the slider SV″. The condense lenses 15, 16 and19 do not need to be attached to the slider SV″. For example, one of thecondense lenses 15, 16 and 19 may be fixed at a predetermined point inan optical axis of the main body of the microscope, except for the othercondense lenses.

4th Embodiment

FIG. 12 illustrates a schematic structure of a fourth embodiment of thepresent invention, and the same portions as those seen in FIG. 3 areindicated by the same reference numerals.

In the case shown in FIG. 12, a point 04 is set in the optical axis 31of the observation optical system light path, between the object lens 2and the point O1 in the structure of FIG. 4, the cube CV1 including thedichoric mirror DM1 is provided so that the point 04 can be a reflectionpoint of the optical axis, and the relay lens 22, collector lens 21 andlaser light source 20 are provided in a common straight line in anoptical axis 35 of the fourth reflected illumination optical systemlight path reflected by the dichroic mirror DM1. The other portions ofare the same as FIG. 4.

In the fourth embodiment thus constituted , if an N₂ laser light sourcehaving a wavelength of 337 nm for the Caged reagent release as the laserlight source 20, the laser light oscillation by the laser light source20 is reflected upward by the characteristics of the dichroic mirror DM1after passing through the collector lens 12 and the relay lens 22, andilluminates a predetermined position in the observation visual field ofthe sample 1.

With this structure, the Caged reagent release can be added to thefluorescence observation and the laser trap, and three light sources canbe used simultaneously by taking advantage of the characteristics of thewavelength of each optical element described in the first embodiment.

With these operations, it is possible to irradiate the excitation light,irradiate the N₂ laser beam onto arbitrary protein in cells whileobserving the fluorescence inside cells to release the Caged reagent,irradiate the IR laser onto the activated protein and optically captureit, and measure a tiny force, which is very effective in the cellphysiological field analyzing the movement of intracellular substancesat the molecular level. That is, various light sources need often to beused simultaneously in pursuit in such a technical field, but thepresent invention can provide an apparatus having large academic meritsdeveloped above, with the structure shown in FIG. 12.

In the fourth embodiment, the dichroic mirror DM2′ for the laser stripis provided to be the lowermost one, of the dichroic mirrors in theother optical axis, but may be provided at any position. In addition,dichroic mirrors in the optical axes can be increased. The cube CV1 isprovided as a single cube, but a plurality of cubes can be inserted ordetached at the position of the cube CV1 by any mechanism.

An inverted-type microscope has been described in the above explanationof the fourth embodiment, but the present invention can be applied to ageneral erecting-type microscope. The kind, wavelength, number andarrangement of the light sources used at this time may not be the same.The crossing angle between each illumination optical system light pathand the observation optical system light path (optical axis 31) does notneed to be set at 90 degrees, and the angles may be different from oneanother. Further, the characteristics of the dichroic mirrors used atthis time may not be in the same, and a dichroic prism or other suitablewavelength classifying means can be applied if it is suitable todesirable luminous flux wavelength characteristics. The mechanism ofinserting the dichroic mirror into the optical axis or detaching ittherefrom may not be the same as the cube turret CT or the slider SV,and any other mechanism can be used. One of the condense lenses 15 and16 may be fixed at a predetermined point in an optical axis of the mainbody of the microscope, without attaching both of the condense lenses 15and 16 to the slider SV.

5th Embodiment

Each of the embodiments can be applied to an erecting-type microscope asdescribed above. An embodiment of the present invention constituted withan erecting-type microscope will be described now with reference to FIG.13.

FIG. 13 illuminates a schematic structure of an embodiment of thepresent invention constituted with an erecting-type microscope. In thefigure, reference numeral 1 denotes a sample to be observed, and areflected illumination and observation optical system including anobject lens is provided over the sample.

The reference symbols attached to the constituent elements of therelated illumination optical system between the object lens 2 and thecondense lens 15 correspond to the reference symbols of the constituentelements shown in FIG. 3. That is, the reflected illuminates opticalsystem of the inverted-type microscope of FIG. 3 described in the firstembodiment is reversed in FIG. 13.

A light path splitting prism Pr is provided over the condense lens 15.The observation light transmitted through the light path splitting prismPr forms an image on the photographing surface Ph of a photographingapparatus, a TV camera, etc. On the other hand, the observation lightreflected at the light path splitting prism Pr forms an image before aneyepiece Oc so that the image can be visually observed through theeyepiece Oc.

The operations of the embodiment thus constituted are quite the same asthose described in the first embodiment. That is, the same advantage canbe also obtained from an erecting-type microscope.

As described in detail, the present invention can provide a reflectedfluorescence microscope rich in usability in a general purpose, having aplurality of independent light paths of the reflected illuminationoptical system, wherein the use of each reflected illumination lightpath is not limited by selecting the reflected illumination light pathsand combining the laser light sources for the Caged reagent release,mercury lamps for excitation light illumination, the laser light sourcesfor the laser trap, etc.

Additional advantages and modifications will readily occur to thoseskilled in the art. Therefore, the invention in its broader aspects isnot limited to the specific details and representative embodiments shownand described herein. Accordingly, various modifications may be madewithout departing from the spirit or scope of the general inventiveconcept as defined by the appended claims and their equivalents.

1. A reflected fluorescence microscope for allowing a plurality ofreflected illumination light beams to be selectively irradiated onto asample, to carry out optical operations of said sample and itsfluorescence observation simultaneously, said microscope comprising: afirst reflected illumination optical system light path provided in adirection which crosses an observation optical system light path to saidsample at a point of intersection; a second reflected illuminationoptical system light path provided in a direction which crosses saidobservation optical system light path at the same point of intersectionas that between said observation optical system light path and saidfirst reflected illumination optical system light path; a first movablemember having a cube including a first dichroic mirror and a cubeincluding a second dichroic mirror, for providing a desired one of saidcubes to be inserted or detached at said point of intersection betweenand observation optical system light path and said first or secondreflected illumination optical system light path; a third reflectedillumination optical system light path provided in a direction whichcrosses said observation optical system light path at a different pointof intersection from that between said first or second reflectedillumination optical system light path; and a second movable memberhaving a cube including a third dichroic mirror, for providing said cubewith said third dichroic mirror to be inserted or detached at said pointof intersection between said observation optical system light path andsaid third reflected illumination optical system light path, whereinsaid first dichroic mirror reflects the light from said first reflectedillumination optical system light path to a side of said sample, whiletransmitting fluorescence emitted from said sample.
 2. A microscopeaccording to claim 1, wherein said second dichroic mirror reflects thelight from said second reflected illumination optical system light pathto a side of said sample, while transmitting fluorescence emitted fromsaid sample.
 3. A microscope according to claim 2, wherein said thirddichroic mirror reflects the light from said third reflectedillumination optical system light path to a side of said sample, whiletransmitting fluorescence emitted from said sample.
 4. A microscopeaccording to claim 1, wherein said first movable member comprises a cubeturret.
 5. A microscope according to claim 1, wherein said secondmovable member comprises a slider.
 6. A microscope according to claim 1,further comprising a laser light source which forms said first reflectedillumination optical system light path.
 7. A microscope according toclaim 6, further comprising a mercury light source which forms saidsecond reflected illumination optical system light path.
 8. A microscopeaccording to claim 7, further comprising at least one of a laser lightsource and a mercury light source which forms said third reflectedillumination optical system light path.
 9. A reflected fluorescencemicroscope for allowing a plurality of reflected illumination lightbeams to be selectively irradiated onto a sample, to carry out opticaloperations of said sample and its fluorescence observationsimultaneously, said microscope comprising: a first reflectedillumination optical system light path provided in a direction whichcrosses an observation optical system light path to said sample at apoint of intersection; a second reflected illumination optical systemlight path provided in a direction which crosses said observationoptical system light path at the same point of intersection as thatbetween said observation optical system light path and said firstreflected illumination optical system light path; a first movable memberhaving a cube including a first dichroic mirror and a cube including asecond dichroic mirror, for providing a desired one of said cubes to beinserted or detached at said point of intersection between saidobservation optical system light path and said first or second reflectedillumination optical system light path; a third reflected illuminationoptical system light path provided in a direction which crosses saidobservation optical system light path at a different point ofintersection from that between said first or second reflectedillumination optical system light path; a second movable member having acube including a third dichroic mirror, for providing said cube withsaid third dichroic mirror to be inserted or detached at said point ofintersection between said observation optical system light path and saidthird reflected illumination optical system light path; and a fourthreflected illumination optical system light path provided in a directionwhich crosses said third reflected illumination optical system lightpath at a point of intersection.
 10. A microscope according to claim 9,further comprising a slider for providing a total reflection mirror tobe inserted or detached at a point of intersection between said thirdand fourth reflected illumination optical system light paths.
 11. Areflected fluorescence microscope for allowing a plurality of reflectedillumination light beams to be selectively irradiated onto a sample, tocarry and carrying out optical operations of said sample and itsfluorescence observation simultaneously, said microscope comprising: anobservation optical system light path which passes through an objectivelens from the sample to an image forming element for forming an image ofthe sample: a first reflected illumination optical system light pathwhich crosses said observation optical system light oath path at a pointof intersection; and a second reflected illumination optical systemlight path which crosses said observation optical system light path atthe same said point of intersection as that between said observationoptical system light path and in a different direction from said firstreflected illumination optical system light path, wherein said firstreflected illumination optical system light path and said secondreflected illumination optical system light path transmit illuminationlight beams differing in spectral distributions.
 12. A microscopeaccording to claim 11, further comprising: a light path splitting memberprovided at a point on said observation optical system light pathbetween said objective lens and said image forming element, and whichsplits light into split light paths; and an eyepiece provided at a pointon a light path which is one of the split light paths split by saidlight path splitting member.
 13. A microscope according to claim 11,further comprising a third related illumination optical system lightpath which crosses said observation optical system light path at adifferent point of intersection from that of said first or secondreflected illumination optical system light path.
 14. A microscopeaccording to claim 13, further comprising wavelength discriminatingmeans provided to be inserted or detected at said point of intersectionbetween said observation optical system light path and said thirdreflected illumination optical system light path.
 15. A microscopeaccording to claim 13, further comprising a fourth reflectedillumination optical system light path which crosses said observationoptical system light path at a different point of intersection from thatof said first, second or third reflected illumination optical systemlight path.
 16. A microscope according to claim 13, further comprising aslider having a cube including a dichroic mirror, for providing saidcube to be inserted or detached at said point of intersection betweensaid observation optical system light path and said third reflectedillumination optical system light path.
 17. A microscope according toclaim 13, further comprising at least one of a laser light source and amercury light source which forms said third reflected illuminationoptical system light path.
 18. A microscope according to claim 11,further comprising a cube turret having a cube including a firstdichroic mirror and a cube including a second dichroic mirror, forproviding a desired one of said cubes to be inserted or detached of saidpoint of intersection between said observation optical system light pathand said first or second reflected illumination optical system lightpath.
 19. A microscope according to claim 11, further comprising a laserlight source which forms said first reflected illumination opticalsystem light path.
 20. A microscope according to claim 19, furthercomprising a mercury light source which forms said second reflectedillumination optical system light path.
 21. A reflected fluorescencemicroscope for allowing a plurality of reflected illumination lightbeams to be selectively irradiated onto a sample, and carrying outoptical operations of said sample and its fluorescence observationsimultaneously, said microscope comprising: an observation opticalsystem light path which passes through an objective lens from the sampleto an image forming element for forming an image of the sample; a firstreflected illumination optical system light path which is formed by afirst light source and which crosses said observation optical systemlight path at a first point of intersection; a second reflectedillumination optical system light path which is formed by a second lightsource and which crosses said observation optical system light path at asecond point of intersection different from the first point ofintersection, the second point of intersection being located betweensaid objective lens and the first point of intersection; and means forinhibiting and allowing transmission of a light beam from the secondlight source of said second reflected illumination to said observationoptical system light path; wherein a wavelength of a light beam from thefirst light source of said first reflected illumination optical systemlight path is longer than a wavelength of a light beam from the secondlight source of said second reflected illumination optical system.
 22. Areflected fluorescence microscope for allowing a plurality of reflectedillumination light beams to be selectively irradiated onto a sample, andcarrying out optical operations of said sample and its fluorescenceobservation simultaneously, said microscope comprising: an observationoptical system light path which passes through an objective lens fromthe sample to an image forming element for forming an image of thesample; a first reflected illumination optical system light path whichis formed by a first light source and which crosses said observationoptical system light path at a first point of intersection; a secondreflected illumination optical system light path which is formed by asecond light source and which crosses said observation optical systemlight path at a second point of intersection different from the firstpoint of intersection, the second point of intersection being locatedbetween said objective lens and the first point of intersection; andmeans for inhibiting and allowing transmission of a light beam from thesecond light source of said second reflected illumination to saidobservation optical system light path; wherein said means for inhibitingand allowing transmission of a light beam from the second light sourcecomprises a cube which includes a dichroic mirror and which is capableof being inserted to and detached from said observation optical systemlight path at the second point of intersection.
 23. A reflectedfluorescence microscope for allowing a plurality of reflectedillumination light beams to be selectively irradiated onto a sample, andcarrying out optical operations of said sample and its fluorescenceobservation simultaneously, said microscope comprising: an observationoptical system light path which passes through an objective lens fromthe sample to an image forming element for forming an image of thesample; a first reflected illumination optical sample light path whichis formed by a first light source and which crosses said observationoptical system light path at a first point of intersection; a secondreflected illumination optical system light path which is formed by asecond light source and which crosses said observation optical systemlight path at a second point of intersection different from the firstpoint of intersection, the second point of intersection being locatedbetween said objective lens and the first point of intersection; andmeans for inhibiting and allowing transmission of a light beam from thesecond light source of said second reflected illumination to saidobservation optical system light path; wherein said means for inhibitingand allowing transmission of a light beam from the second light sourcecomprises a plurality of cubes, each of which is capable of beinginserted to and detached from said observation optical system light pathat the second point of intersection.
 24. A reflected fluorescencemicroscope for allowing a plurality of reflected illumination lightbeams to be selectively irradiated onto a sample, and carrying outoptical operations of said sample and its fluorescence observationsimultaneously, said microscope comprising: an observation opticalsystem light path which passes through an objective lens from the sampleto an image forming element for forming an image of the sample; a firstreflected illumination optical system light path which is formed by afirst light source and which crosses said observation optical systemlight path at a first point of intersection; a section reflectedillumination optical system light path which is formed by a second lightsource and which crosses said observation optical system light path at asecond point of intersection different from the first point ofintersection, the second point of intersection being located betweensaid objective lens and the first point of intersection; means forinhibiting and allowing transmission of a light beam from the secondlight source of said second reflected illumination to said observationoptical system light path; and a slider having a cube including adichroic mirror, and wherein said slider is adapted to insert and removesaid cube from said observation optical system light path at the firstpoint of intersection.
 25. A reflected fluorescence microscope forallowing a plurality of reflected illumination light beams to beselectively irradiated onto a sample, and carrying out opticaloperations of said sample and its fluorescence observationsimultaneously, said microscope comprising: an observation opticalsystem light path which passes through an objective lens from the sampleto an image forming element for forming an image of the sample; a firstreflected illumination optical system light path which is formed by afirst light source and which crosses said observation optical systemlight path at a first point of intersection; a second reflectedillumination optical system light path which is formed by a second lightsource and which crosses said observation optical system light path at asecond point of intersection different from the first point ofintersection, the second point of intersection being located betweensaid objective lens and the first point of intersection; and means forinhibiting and allowing transmission of a light beam from the secondlight source of said second reflected illumination to said observationoptical system light path; wherein said second light source includes atleast one of a laser light source and a mercury light source which formssaid first reflected illumination optical system light path; and whereinsaid second light source produces an N ₂ laser having a wavelength at337 nm.
 26. A reflected fluorescence microscope for allowing a pluralityof reflected illumination light beams to be selectively irradiated ontoa sample, and carrying out optical operations of said sample and itsfluorescence observation simultaneously, said microscope comprising: anobservation optical system light path which passes through an objectivelens from the sample to an image forming element for forming an image ofthe sample; a first reflected illumination optical system light pathwhich is formed by a first light source and which crosses saidobservation optical system light path at a first point of intersection;a second reflected illumination optical system light path which isformed by a second light source and which crosses said observationoptical system light path at a second point of intersection differentfrom the first point of intersection, the second point of intersectionbeing located between said objective lens and the first point ofintersection; and means for inhibiting and allowing transmission of alight beam from the second light source of said second reflectedillumination to said observation optical system light path; wherein awavelength of a light beam from the first light source of said firstreflected illumination optical system light path is different from awavelength of a light beam from the second light source of said secondreflected illumination optical system.
 27. A reflected fluorescencemicroscope for allowing a plurality of reflected illumination lightbeams to be selectively irradiated onto a sample, and carrying outoptical operations of said sample and its fluorescence observationsimultaneously, said microscope comprising: an observation opticalsystem light path which passes through an objective lens from the sampleto an image forming element for forming an image of the sample; a firstreflected illumination optical system light path which is formed by afirst light source and which crosses said observation optical systemlight path at a first point of intersection; a second reflectedillumination optical system light path which is formed by a second lightsource and which crosses said observation optical system light path at asecond point of intersection different from the first point ofintersection, the second point of intersection being located betweensaid objective lens and the first point of intersection; and means forinhibiting and allowing transmission of a light beam from the secondlight source of said second reflected illumination to said observationoptical system light path; wherein said means for inhibiting andallowing transmission of a light beam from the second light sourcecomprises a cube which includes a dichroic mirror and which is capableof being inserted to and detached from said observation optical systemlight path at the second point of intersection.
 28. A microscopeaccording to claim 27, wherein a wavelength of a light beam from thefirst light source of said first reflected illumination optical systemlight path is different from a wavelength of a light beam from thesecond light source of said second reflected illumination opticalsystem.
 29. A microscope according to claim 27, wherein a wavelength ofa light beam from the first light source of said first reflectedillumination optical system light path is longer than a wavelength of alight beam from the second light source of said second reflectedillumination optical system.
 30. A microscope according to claim 27,wherein said means for inhibiting and allowing transmission of a lightbeam from the second light source comprises a plurality of cubes, eachof which is capable of being inserted to and detached from saidobservation optical system light path at the second point ofintersection.
 31. A microscope according to claim 27, further comprisinga light path splitting member which is provided at a point on saidobservation optical system light path between said objective lens andsaid image forming element, and which splits light into two split lightpaths.
 32. A microscope according to claim 27, further comprising aslider having a cube including a dichroic mirror, and wherein saidslider is adapted to insert and remove said cube from said observationoptical system light path at the first point of intersection.
 33. Amicroscope according to claim 27, wherein said second light sourceincludes at least one of a laser light source and a mercury light sourcewhich forms said first reflected illumination optical system light path.34. A microscope according to claim 33, wherein said second light sourceproduces an N₂ laser having a wavelength of 337 nm.